Metagenomic insights into the wastewater resistome before and after purification at large­scale wastewater treatment plants in the Moscow city.

Wastewater treatment plants (WWTPs) are considered to be hotspots for the spread of antibiotic resistance genes (ARGs). We performed a metagenomic analysis of the raw wastewater, activated sludge and treated wastewater from two large WWTPs responsible for the treatment of urban wastewater in Moscow, Russia. In untreated wastewater, several hundred ARGs that could confer resistance to most commonly used classes of antibiotics were found. WWTPs employed a nitrification/denitrification or an anaerobic/anoxic/oxic process and enabled efficient removal of organic matter, nitrogen and phosphorus, as well as fecal microbiota. The resistome constituted about 0.05% of the whole metagenome, and after water treatment its share decreased by 3-4 times. The resistomes were dominated by ARGs encoding resistance to beta-lactams, macrolides, aminoglycosides, tetracyclines, quaternary ammonium compounds, and sulfonamides. ARGs for macrolides and tetracyclines were removed more efficiently than beta-lactamases, especially ampC, the most abundant ARG in the treated effluent. The removal efficiency of particular ARGs was impacted by the treatment technology. Metagenome-assembled genomes of multidrug-resistant strains were assembled both for the influent and the treated effluent. Ccomparison of resistomes from WWTPs in Moscow and around the world suggested that the abundance and content of ARGs depend on social, economic, medical, and environmental factors.

Fecal Microbiota and Diet Composition of Buryatian Horses Grazing Warm- and Cold-Season Grass Pastures.

The Buryatian horse is an ancient breed and, as an indigenous breed, they have unique adaptive abilities to use scarce pastures, graze in winter, and survive in harsh conditions with minimal human care. In this study, fecal microbiota of Buryatian horses grazing in the warm and cold seasons were investigated using NGS technology on the Illumina MiSeq platform. We hypothesized that the composition of microbial communities in the feces of horses maintained on pasture would change in the different seasons, depending on the grass availability and different plant diets. We conducted microhistological fecal studies of horse diet composition on steppe pasture. The alpha diversity analysis showed horses had a more abundant and diverse gut microbiota in summer. There were significant effects on the beta diversity of microbial families, which were clustered by the warm and cold season in a principal coordinate analysis (PCoA), with 45% of the variation explained by two principal coordinates. This clustering by season was further confirmed by the significant differences observed in the relative abundances of microbial families and genera. The obtained results can serve as an experimental substantiation for further study of the impact of pasture grasses, which have a pharmacological effect, on the diversity of the gut microbiome and horse health.

Active Sulfate-Reducing Bacterial Community in the Camel Gut.

The diversity and activity of sulfate-reducing bacteria (SRB) in the camel gut remains largely unexplored. An abundant SRB community has been previously revealed in the feces of Bactrian camels (Camelus bactrianus). This study aims to combine the 16S rRNA gene profiling, sulfate reduction rate (SRR) measurement with a radioactive tracer, and targeted cultivation to shed light on SRB activity in the camel gut. Fresh feces of 55 domestic Bactrian camels grazing freely on semi-arid mountain pastures in the Kosh-Agach district of the Russian Altai area were analyzed. Feces were sampled in early winter at an ambient temperature of -15 °C, which prevented possible contamination. SRR values measured with a radioactive tracer in feces were relatively high and ranged from 0.018 to 0.168 nmol S cm-3 day-1. The 16S rRNA gene profiles revealed the presence of Gram-negative Desulfovibrionaceae and spore-forming Desulfotomaculaceae. Targeted isolation allowed us to obtain four pure culture isolates belonging to Desulfovibrio and Desulforamulus. An active SRB community may affect the iron and copper availability in the camel intestine due to metal ions precipitation in the form of sparingly soluble sulfides. The copper-iron sulfide, chalcopyrite (CuFeS2), was detected by X-ray diffraction in 36 out of 55 analyzed camel feces. In semi-arid areas, gypsum, like other evaporite sulfates, can be used as a solid-phase electron acceptor for sulfate reduction in the camel gastrointestinal tract.

Distribution Patterns of Antibiotic Resistance Genes and Their Bacterial Hosts in a Manure Lagoon of a Large-Scale Swine Finishing Facility.

The spread of antibiotic resistance genes (ARGs) that are present in livestock manures, which are discharged into the environment, is a severe threat to human and animal health. Here, we used 16S rRNA gene profiling and metagenomic analysis to characterize microbial community composition and antibiotic resistance in a manure storage lagoon from a large-scale swine finishing facility. Manure samples were collected at intervals of two years. Both the prokaryotic community and the resistome were dominated by the Firmicutes, Proteobacteria and Bacteroidota. Metagenomic analysis of two samples revealed 726 and 641 ARGs classified into 59 and 46 AMR gene families. Besides multidrug efflux pumps, the predominating ARGs potentially encoded resistance to tetracyclines, macrolide-lincosamide-streptogramin, aminoglycosides, peptide antibiotics, rifamycin, chloramphenicol, and beta-lactams. Genes from all predominant AMR gene families were found in both samples indicating overall long-term stability of the resistome. Antibiotic efflux pumps were the primary type of ARGs in the Proteobacteria, while antibiotic target alteration or protection was the main mechanism of resistance in the Firmicutes, Actinobacteriota and Bacteroidota. Metagenome-assembled genomes (MAG) of four multidrug-resistant strains were assembled. The first MAG, assigned to Escherichia flexneri, contained 46 ARGs, including multidrug efflux pumps, modified porins, beta-lactamases, and genes conferring resistance to peptide antibiotics. The second MAG, assigned to the family Alcaligenaceae, contained 18 ARGs encoding resistance to macrolide-lincosamide-streptogramin, tetracyclines, aminoglycosides and diaminopyrimidins. Two other MAGs representing the genera Atopostipes and Prevotella, contained four and seven ARGs, respectively. All these MAGs represented minor community members and accounted for less than 0.3% of the whole metagenome. Overall, a few lineages originated from the gut but relatively rare in the manure storage lagoon, are the main source of ARGs and some of them carry multiple resistance determinants.

Genome analysis of the candidate phylum MBNT15 bacterium from a boreal peatland predicted its respiratory versatility and dissimilatory iron metabolism.

Uncultured bacteria of the candidate phylum MBNT15, distantly related to Desulfobacterota, have been identified in a broad range of mostly organic-rich aquatic environments. We assembled a near-complete genome of a member of MBNT15 from a boreal peatland metagenome and used genomic data to analyze the metabolic pathways of this bacterium and its ecological role. This bacterium, designated SHF-111, was predicted to be rod shaped, it lacks flagellar machinery but twitching motility is encoded. Genome-based phylogenetic analysis supported the phylum-level classification of the MBNT15 lineage. Genome annotation and metabolic reconstruction revealed the presence of the Embden-Meyerhof, Entner-Doudoroff and pentose phosphate pathways, as well as the complete tricarboxylic acid (TCA) cycle, and suggested a facultatively anaerobic chemoheterotrophic lifestyle with the ability to ferment peptides, amino acids, fatty acids and simple sugars, and completely oxidize these substrates through aerobic and anaerobic respiration. The SHF-111 genome encodes multiple multiheme c-type cytochromes that probably enable dissimilatory iron reduction. Consistently, the relative abundance of MBNT15 in peatlands positively correlated with iron concentration. Apparently, in the wetland ecosystem, MBNT15 representatives play the role of scavengers, carrying out the complete mineralization of low molecular weight organic substances formed as a result of microbial degradation of complex polymeric substrates. Comparative genome analysis of the MBNT15 phylum revealed that vast majority of its members are capable of aerobic respiration and dissimilatory iron reduction and some species also can reduce sulfur and nitrogen compounds, but not sulfate. Based on phylogenetic and genomic analyses, the novel bacterium is proposed to be classified as Candidatus Deferrimicrobium borealis, within a candidate phylum Deferrimicrobiota.

The structure of microbial communities of activated sludge of large-scale wastewater treatment plants in the city of Moscow.

Microbial communities in wastewater treatment plants (WWTPs) play a key role in water purification. Microbial communities of activated sludge (AS) vary extensively based on plant operating technology, influent characteristics and WWTP capacity. In this study we performed 16S rRNA gene profiling of AS at nine large-scale WWTPs responsible for the treatment of municipal sewage from the city of Moscow, Russia. Two plants employed conventional aerobic process, one plant-nitrification/denitrification technology, and six plants were operated with the University of Cape Town (UCT) anaerobic/anoxic/oxic process. Microbial communities were impacted by the technology and dominated by the Proteobacteria, Bacteroidota and Actinobacteriota. WWTPs employing the UCT process enabled efficient removal of not only organic matter, but also nitrogen and phosphorus, consistently with the high content of ammonia-oxidizing Nitrosomonas sp. and phosphate-accumulating bacteria. The latter group was represented by Candidatus Accumulibacter, Tetrasphaera sp. and denitrifiers. Co-occurrence network analysis provided information on key hub microorganisms in AS, which may be targeted for manipulating the AS stability and performance. Comparison of AS communities from WWTPs in Moscow and worldwide revealed that Moscow samples clustered together indicating that influent characteristics, related to social, cultural and environmental factors, could be more important than a plant operating technology.

Highly Distinct Microbial Communities in Elevated Strings and Submerged Flarks in the Boreal Aapa-Type Mire.

Large areas in the northern hemisphere are covered by extensive wetlands, which represent a complex mosaic of raised bogs, eutrophic fens, and aapa mires all in proximity to each other. Aapa mires differ from other types of wetlands by their concave surface, heavily watered by the central part, as well as by the presence of large-patterned string-flark complexes. In this paper, we characterized microbial diversity patterns in the surface peat layers of the neighboring string and flark structures located within the mire site in the Vologda region of European North Russia, using 16S rRNA gene sequencing. The microbial communities in raised strings were clearly distinct from those in submerged flarks. Strings were dominated by the Alpha- and Gammaproteobacteria. Other abundant groups were the Acidobacteriota, Bacteroidota, Verrucomicrobiota, Actinobacteriota, and Planctomycetota. Archaea accounted for only 0.4% of 16S rRNA gene sequences retrieved from strings. By contrast, they comprised about 22% of all sequences in submerged flarks and mostly belonged to methanogenic lineages. Methanotrophs were nearly absent. Other flark-specific microorganisms included the phyla Chloroflexi, Spirochaetota, Desulfobacterota, Beijerinckiaceae- and Rhodomicrobiaceae-affiliated Alphaproteobacteria, and uncultivated groups env.OPS_17 and vadinHA17 of the Bacteroidota. Such pattern probably reflects local anaerobic conditions in the submerged peat layers in flarks.

Peat-Inhabiting Verrucomicrobia of the Order Methylacidiphilales Do Not Possess Methanotrophic Capabilities.

Methanotrophic verrucomicrobia of the order Methylacidiphilales are known as extremely acidophilic, thermophilic or mesophilic bacteria that inhabit acidic geothermal ecosystems. The occurrence of verrucomicrobial methanotrophs in other types of acidic environments remains an open question. Notably, Methylacidiphilales-affiliated 16S rRNA gene sequences are commonly retrieved from acidic (pH 3.5-5.5) peatlands. In this study, we compared the patterns of verrucomicrobial diversity in four acidic raised bogs and six neutral fens located in European North Russia. Methylacidiphilales-like 16S rRNA gene reads displaying 83-86% similarity to 16S rRNA gene sequences of currently described verrucomicrobial methanotrophs were recovered exclusively from raised bogs. Laboratory incubation of peat samples with 10% methane for 3 weeks resulted in the pronounced increase of a relative abundance of alphaproteobacterial methanotrophs, while no response was detected for Methylacidiphilales-affiliated bacteria. Three metagenome-assembled genomes (MAGs) of peat-inhabiting Methylacidiphilales bacteria were reconstructed and examined for the presence of genes encoding methane monooxygenase enzymes and autotrophic carbon fixation pathways. None of these genomic determinants were detected in assembled MAGs. Metabolic reconstructions predicted a heterotrophic metabolism, with a potential to hydrolyze several plant-derived polysaccharides. As suggested by our analysis, peat-inhabiting representatives of the Methylacidiphilales are acidophilic aerobic heterotrophs, which comprise a sister family of the methanotrophic Methylacidiphilaceae.

Microbial Communities Involved in Methane, Sulfur, and Nitrogen Cycling in the Sediments of the Barents Sea.

A combination of physicochemical and radiotracer analysis, high-throughput sequencing of the 16S rRNA, and particulate methane monooxygenase subunit A (pmoA) genes was used to link a microbial community profile with methane, sulfur, and nitrogen cycling processes. The objects of study were surface sediments sampled at five stations in the northern part of the Barents Sea. The methane content in the upper layers (0-5 cm) ranged from 0.2 to 2.4 µM and increased with depth (16-19 cm) to 9.5 µM. The rate of methane oxidation in the oxic upper layers varied from 2 to 23 nmol CH4 L-1 day-1 and decreased to 0.3 nmol L-1 day-1 in the anoxic zone at a depth of 16-19 cm. Sulfate reduction rates were much higher, from 0.3 to 2.8 µmol L-1 day-1. In the surface sediments, ammonia-oxidizing Nitrosopumilaceae were abundant; the subsequent oxidation of nitrite to nitrate can be carried out by Nitrospira sp. Aerobic methane oxidation could be performed by uncultured deep-sea cluster 3 of gamma-proteobacterial methanotrophs. Undetectable low levels of methanogenesis were consistent with a near complete absence of methanogens. Anaerobic methane oxidation in the deeper sediments was likely performed by ANME-2a-2b and ANME-2c archaea in consortium with sulfate-reducing Desulfobacterota. Sulfide can be oxidized by nitrate-reducing Sulfurovum sp. Thus, the sulfur cycle was linked with the anaerobic oxidation of methane and the nitrogen cycle, which included the oxidation of ammonium to nitrate in the oxic zone and denitrification coupled to the oxidation of sulfide in the deeper sediments. Methane concentrations and rates of microbial biogeochemical processes in sediments in the northern part of the Barents Sea were noticeably higher than in oligotrophic areas of the Arctic Ocean, indicating that an increase in methane concentration significantly activates microbial processes.